The present invention relates to chemically modified subtilisin proteases which are useful in compositions such as, for example, personal care compositions, laundry compositions, hard surface cleansing compositions, and light duty cleaning compositions.
Enzymes make up the largest class of naturally occurring proteins. One class of enzyme includes proteases which catalyze the hydrolysis of other proteins. This ability to hydrolyze proteins has typically been exploited by incorporating naturally occurring and genetically engineered proteases into cleaning compositions, particularly those relevant to laundry applications.
In the cleaning arts, the mostly widely utilized of these proteases are the serine proteases. Most of these serine proteases are produced by bacterial organisms while some are produced by other organisms, such as fungi. See Siezen et al., xe2x80x9cHomology Modelling and Protein Engineering Strategy of Subtilases, the Family of Subtilisin-Like Serine Proteasesxe2x80x9d, Protein Engineering, Vol. 4, No. 7, pp. 719-737 (1991). Unfortunately, the efficacy of the wild-type proteases in their natural environment is frequently not optimized for the artificial environment of a cleaning composition. Specifically, protease characteristics such as, for example, thermal stability, pH stability, oxidative stability, and substrate specificity are not necessarily optimized for utilization outside the natural environment of the protease.
Several approaches have been employed to alter the wild-type amino acid sequence of serine proteases with the goal of increasing the efficacy of the protease in the unnatural wash environment. These approaches include the genetic redesign and/or chemical modification of proteases to enhance thermal stability and to improve oxidation stability under quite diverse conditions.
However, because such modified proteases are foreign to mammals, they are potential antigens. As antigens, these proteases cause an immunogenic and/or allergenic response (herein collectively described as immunogenic response) in mammals.
Furthermore, while genetic redesign and chemical modification of proteases has been prominent in the continuing search for more highly effective proteases for laundry applications, such proteases have not been commercially utilized in personal care compositions and light duty detergents. A primary reason for the absence of these proteases in products such as, for example, soaps, gels, body washes, shampoos, and light duty dish detergents is due to the problem of human sensitization leading to undesirable immunogenic responses. It would therefore be highly advantageous to provide a personal care composition or a light duty detergent which provides the cleansing properties of proteases without the provocation of an immunogenic response.
Presently, immunogenic response to proteases may be minimized by immobilizing, granulating, coating, or dissolving chemically modified proteases to avoid their becoming airborne. These methods, while addressing consumer exposure to airborne proteases, still present the risks associated with extended tissue contact with the finished composition.
It has also been proposed that reduction in immunogenicity of a protease may be achieved by attaching polymers to the protease. See. e.g., U.S. Pat. No. 4,179,337, Davis et al., issued Dec. 18, 1979; U.S. Pat. No. 5,856,451, Olsen et al., assigned to Novo Nordisk, issued Jan. 5, 1999; WO 99/00489, Olsen et al., assigned to Novo Nordisk, published Jan. 7, 1999; WO 98/30682, Olsen et al., assigned to Novo Nordisk, published Jul. 16, 1998; and WO 98/35026, Von Der Osten et al., published Aug. 13, 1998. However, such proposals have not suggested the importance of attaching polymers to particular amino acid regions of the protease in order to most effectively decrease the immunogenic response.
It has recently been discovered that the subtilisin protease comprises three epitope regions and that conjugation of one or more polymers, polypeptides, or other groups should be attached at one or more of these regions to effect significant reduction in immunogenicity of the protease. See, e.g., U.S. patent application Ser. No. 09/088,912, Weisgerber et al., assigned to The Procter and Gamble Co., filed Jun. 2, 1998.
As an alternative to protection of the epitope regions of the subtilisin protease, the present inventors have discovered that steric protection of one or more xe2x80x9cclip sitesxe2x80x9d (i.e., locations of the protease where hydrolysis occurs in vivo) of the protease may be utilized to prevent or impede presentation of an epitope and decrease the immunogenicity of the protease. Accordingly, the present inventors provide chemically modified subtilisins wherein the chemical modification is at a region in steric proximity to one or more of the clip sites. The present inventors have therefore discovered subtilisin proteases which evoke a decreased immunogenic response yet maintain their activity as an efficient and active protease. Accordingly, the present protease conjugates are suitable for use in several types of compositions including, but not limited to, laundry, dish, hard surface, skin care, hair care, beauty care, oral care, and contact lens compositions.
The present invention relates to protease conjugates comprising a protease moiety and one or more addition moieties wherein each addition moiety is covalently attached to an amino acid of the protease moiety at a position selected from the group consisting of 13, 14, 15, 16, 18, 19, 20, 21, 84, 85, 88, 158, 159, 160, 161, 162, 163, 164, 165, 170, 186, 191, 192, 193, 194, 196, 259, 260, 261, 262, and 274 corresponding to subtilisin BPNxe2x80x2; wherein the addition moieties each, independently, have the structure: 
wherein X is selected from the group consisting of nil and a linking moiety; R1 is selected from the group consisting of nil, a first polypeptide, and a first polymer; and R2 is selected from the group consisting of nil, a second polypeptide, and a second polymer; wherein at least one of X, R1, and R2 is not nil.
The protease conjugates of the present invention have decreased immunogenicity relative to the parent protease. Accordingly, such protease conjugates are suitable for use in several types of compositions including, but not limited to, laundry, dish, hard surface, skin care, hair care, beauty care, oral care, and contact lens compositions.
The essential components of the present invention are herein described below. Also included are non-limiting descriptions of various optional and preferred components useful in embodiments of the present invention.
The present invention can comprise, consist of, or consist essentially of any of the required or optional components and/or limitations described herein.
All percentages and ratios are calculated by weight unless otherwise indicated. All percentages are calculated based on the total composition unless otherwise indicated.
All component or composition levels are in reference to the active level of that component or composition, and are exclusive of impurities, for example, residual solvents or by-products, which may be present in commercially available sources.
All documents referred to herein, including all patents, patent applications, and publications, are hereby incorporated by reference in their entirety.
Referred to herein are trade names for materials including, but not limited to, enzymes. The inventors herein do not intend to be limited by materials under a certain trade name. Equivalent materials (e.g., those obtained from a different source under a different name or catalog (reference) number) to those referenced by trade name may be substituted and utilized in the protease conjugates and compositions herein.
As used herein, abbreviations will be used to describe amino acids. Table I provides a list of abbreviations used herein:
As used herein, the term xe2x80x9cmutationxe2x80x9d refers to an alteration in a gene sequence and/or an amino acid sequence produced by those gene sequences. Mutations include deletions, substitutions, and additions of amino acid residues to the wild-type protein sequence.
As used herein, the term xe2x80x9cparentxe2x80x9d refers to a protein (wild-type or variant) which is utilized for further modification to form a protease conjugate herein.
As used herein, the term xe2x80x9cwild-typexe2x80x9d refers to a protein, for example a protease or other enzyme, produced by a naturally-occurring organism.
As used herein, the term xe2x80x9cvariantxe2x80x9d means a protein having an amino acid sequence which differs from that of the corresponding wild-type protein.
As used herein, all polymer molecular weights are expressed as weight average molecular weights.
As referred to herein, while the conjugates of the present invention are not limited to those comprising subtilisin BPNxe2x80x2 and variants thereof, all amino acid numbering is with reference to the amino acid sequence for subtilisin BPNxe2x80x2 which is represented by SEQ ID NO:1. The amino acid sequence for subtilisin BPNxe2x80x2 is further described by Wells et al., Nucleic Acids Research, Vol. II, pp. 7911-7925 (1983).
The protease conjugates of the present invention are compounds which comprise a protease moiety and one or more addition moieties, wherein the protease moiety and the addition moieties are connected via covalent attachment (i.e., covalent bonding).
Protease Moieties
The protease moieties herein are subtilisin-like proteases, either wild-type or variants thereof. As used herein, the term xe2x80x9csubtilisin-like proteasexe2x80x9d means a protease which has at least 50%, and preferably 80%, amino acid sequence identity with the sequences of subtilisin BPNxe2x80x2. Wild-type subtilisin-like proteases are produced by, for example, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus amylosaccharicus, Bacillus licheniformis, Bacillus lentus, and Bacillus subtilis microorganisms. A discussion relating to subtilisin-like serine proteases and their homologies may be found in Siezen et al., xe2x80x9cHomology Modelling and Protein Engineering Strategy of Subtilases, the Family of Subtilisin-Like Serine Proteasesxe2x80x9d, Protein Engineering, Vol. 4, No. 7, pp. 719-737 (1991).
Preferred protease moieties for use herein include, for example, those obtained from Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus subtilis, subtilisin BPN, subtilisin BPNxe2x80x2, subtilisin Carlsberg, subtilisin DY, subtilisin 309, proteinase K, and thermitase, including A/S Alcalase(copyright) (commercially available from Novo Industries, Copenhagen, Denmark), Esperase(copyright) (Novo Industries), Savinase(copyright) (Novo Industries), Maxatase(copyright) (commercially available from Genencor International Inc.), Maxacal(copyright) (Genencor International Inc.), Maxapem 15(copyright) (Genencor International Inc.), and variants of the foregoing. Especially preferred protease moieties for use herein include those obtained from Bacillus amyloliquefaciens and variants thereof. The most preferred protease moieties herein are subtilisin BPNxe2x80x2 and variants thereof.
Especially preferred variants of subtilisin BPNxe2x80x2, hereinafter referred to as xe2x80x9cProtease Axe2x80x9d, for use herein are disclosed in U.S. Pat. No. 5,030,378, Venegas, issued Jul. 9, 1991, as characterized by the subtilisin BPNxe2x80x2 amino acid sequence with the following mutations:
(a) Gly at position 166 is substituted with an amino acid residue selected from Asn, Ser,. Lys, Arg, His, Gln, Ala and Glu; Gly at position 169 is substituted with Ser; and Met at position 222 is substituted with an amino acid residue selected from Gln, Phe, His, Asn, Glu, Ala and Thr; or
(b) Gly at position 160 is substituted with Ala, and Met at position 222 is substituted with Ala.
Additionally preferred variants of subtilisin BPNxe2x80x2, hereinafter referred to as xe2x80x9cProtease Bxe2x80x9d, for use herein are disclosed in EP 251,446, assigned to Genencor International, Inc., published Jan. 7, 1988, as characterized by the wild-type subtilisin BPNxe2x80x2 amino acid sequence with mutations at one or more of the following positions: Tyr21, Thr22, Ser24, Asp36, Ala45, Ala48, Ser49, Met50, His67, Ser87, Lys94, Val95, Gly97, Ser101, Gly102, Gly103, Ile107, Gly110, Met124, Gly127, Gly128, Pro129, Leu135, Lys170, Tyr171, Pro172, Asp197, Met199, Ser204, Lys213, Tyr214, Gly215, and Ser221; or two or more of the positions listed above combined with one or more mutations at positions selected from Asp32, Ser33, Tyr104, Ala152, Asn155, Glu156, Gly166, Gly169, Phe189, Tyr217, and Met222.
Other preferred subtilisin BPNxe2x80x2 variants for use herein are hereinafter referred to as xe2x80x9cProtease Cxe2x80x9d, and are described in WO 95/10615, assigned to Genencor International Inc., published Apr. 20, 1995, as characterized by the wild-type subtilisin BPNxe2x80x2 amino acid sequence with a mutation to position Asn76, in combination with mutations in one or more other positions selected from Asp99, Ser101, Gln103, Try104, Ser105, Ile107, Asn109, Asn123, Leu126, Gly127, Gly128, Leu135, Glu156, Gly166, Glu195, Asp197, Ser204, Gln206, Pro210, Ala216, Tyr217, Asn218, Met222, Ser260, Lys265, and Ala274.
Other preferred subtilisin BPNxe2x80x2 variants for use herein, hereinafter referred to as xe2x80x9cProtease Dxe2x80x9d, are described in U.S. Pat. No. 4,760,025, Estell et al., issued Jul. 26, 1988, as characterized by the wild-type subtilisin BPNxe2x80x2 amino acid sequence with mutations to one or more amino acid positions selected from the group consisting of Asp32, Ser33, His64, Try104, Asn155, Glu156, Gly166, Gly169, Phe189, Tyr217, and Met222.
The more preferred protease moieties herein are selected from the group consisting of Alcalase(copyright), subtilisin BPNxe2x80x2, Protease A, Protease B, Protease C, and Protease D, with Protease D being the most preferred.
Without intending to be limited by theory, the protease moieties herein have at least two initial xe2x80x9cclip sitesxe2x80x9d, or regions of the protease moiety which are particularly susceptible to in vivo hydrolysis. The region which is most susceptible to in vivo hydrolysis is at amino acid positions 160 through 165, inclusive, corresponding to subtilisin BPNxe2x80x2. Another region susceptible to in vivo hydrolysis is amino acid positions 19 and 20, corresponding to subtilisin BPNxe2x80x2. The present inventors have discovered that these clip sites are protected from hydrolysis, and thus exposure of epitopes, by covalently attaching one or more addition moieties to an amino acid of the protease moiety at a position selected from 13, 14, 15, 16, 18, 19, 20, 21, 84, 85, 88, 158, 159, 160, 161, 162, 163, 164, 165, 170, 186, 191, 192, 193, 194, 196, 259, 260, 261, 262, and 274 corresponding to subtilisin BPNxe2x80x2. Such positions are hereafter collectively referred to as the xe2x80x9cclip site protection positions.xe2x80x9d
Preferably the positions are selected from 13, 14, 15, 16, 18, 19, 20, 21, 158, 159, 160, 161, 162, 163, 164, 165, 170, 186, 191, 192, 193, 194, 196, 259, 260, 261, and 262 corresponding to subtilisin BPNxe2x80x2. More preferably, the positions are selected from 14, 15, 16, 18, 19, 20, 21, 158, 159, 160, 161, 162, 163, 164, 165, 170, 186, 191, 192, 193, 194, 196, 259, 260, 261, and 262 corresponding to subtilisin BPNxe2x80x2. Still more preferably, the positions are selected from 18, 19, 20, 21, 158, 159, 160, 161, 162, 163, 164, 165, 170, 186, 191, 192, 193, 194, 196, 259, 260, 261, and 262 corresponding to subtilisin BPNxe2x80x2. Even more preferably, the positions are selected from 158, 159, 160, 161, 162, 163, 164, 165, 170, 186, 191, 192, 193, 194, 196, 259, 260, 261, and 262 corresponding to subtilisin BPNxe2x80x2. Within this group, the positions are more preferably selected from 158, 159, 160, 161, 162, 163, 164, 165, 170, 191, 192, 193, 194, 261, and 262 corresponding to subtilisin BPNxe2x80x2. More preferably, the positions are selected from 158, 159, 160, 161, 162, 163, 164, 192, 193, 194, 261, and 262 corresponding to subtilisin BPNxe2x80x2. Most preferably, the positions are selected from 160, 161, 162, 163, and 261 corresponding to subtilisin BPNxe2x80x2.
In a preferred embodiment of the present invention, the protease moiety comprises a modified sequence of a parent amino acid sequence. The parent amino acid sequence may be any of the above proteases described above, with the same preferred limitations as described above. In this embodiment, the parent amino acid sequence is substituted at one or more of the parent amino acid residues with a substituting amino acid to produce a protease moiety suitable for attachment with one or more of the present addition moieties. In accordance with the present invention, the substitution should be made at one or more positions at one or more of the clip site protection positions. The clip site protection positions, and preferred limitations thereof, are described above.
In order to best achieve selective attachment at one or more of the clip site protection positions of one or more addition moieties to the protease moiety, the substitution should be with a substituting amino acid which does not occur in (is unique to) the parent amino acid sequence. In this respect, any substituting amino acid which is unique to the parent amino acid sequence may be utilized. For example, because a cysteine residue does not occur in the wild-type amino acid sequence for subtilisin BPNxe2x80x2, a substitution of subtilisin BPNxe2x80x2 with one or more cysteine residues at one or more of the clip site protection positions is suitable for the present invention. Wherein a cysteine residue occurs at a position other than a clip site protection position of the parent amino acid sequence, it is preferable to substitute another amino acid residue for in each of those positions to enable selective coupling with one or more addition moieties at a clip site protection position. Cysteine is the most preferred substituting amino acid for substitution at one or more of the clip site protection positions.
Other preferred substituting amino acids include lysine. Wherein the substituting amino acid is lysine, it is preferred to mutate lysine residues which occur at positions other than a clip site protection position of the parent amino acid sequence to another amino acid residue such that functionalization of one or more of the lysine residues at a clip site protection position is selective. For example, a lysine residue occurs at position 170 of subtilisin BPNxe2x80x2 which is a clip site protection position as defined herein. Site-selective mutation of all other lysine residues occurring in the subtilisin BPNxe2x80x2 sequence may be performed followed by selective functionalization of the lysine residue at position 170 with an addition moiety. Alternatively, amino acid residues at any of the clip site protection positions may be mutated to lysine (for example) followed by selective functionalization at those positions by an addition moiety.
Addition Moieties
The protease conjugates of the present invention comprise one or more addition moieties wherein each of the addition moieties is covalently attached to one of the amino acid residues at a clip site protection position as described herein. The addition moiety may be any chemical structure. Preferably, the addition moiety sterically hinders the clip site protection position to which it is attached, or any other clip site protection position as defined herein. Non-limiting examples of addition moieties include organic molecules including, but not limited to, molecules having a molecular weight of less than about 1600, preferably less than about 800, more preferably less than about 400, and most preferably less than about 300; polypeptides; and polymers. As used herein, the term xe2x80x9cpolypeptidexe2x80x9d means a molecule comprising two or more amino acid residues. As used herein, the term xe2x80x9cpolymerxe2x80x9d means any molecule which comprises two or more identical (preferably five or more identical) monomer units.
Preferably, the addition moiety has the structure: 
wherein X is selected from nil and a linking moiety; R1 is selected from the group consisting of nil, a first polypeptide, and a first polymer; and R2 is selected from the group consisting of nil, a second polypeptide, and a second polymer, wherein at least one of X, R1, and R2 is not nil.
Preferably, the protease conjugate comprises from 1 to about 15, more preferably from about 2 to about 10, and most preferably from about 1 to about 5 addition moieties.
Wherein R1 and R2 are each, independently, polypeptide moieties or polymer moieties, R1 and R2 may be identical or different. Preferably, wherein R1 is a polypeptide moiety, R2 is selected from nil and a polypeptide moiety, and is most preferably nil. Most preferably, wherein R1 is a polypeptide moiety, R2 is selected from nil and an identical polypeptide moiety, and is most preferably nil. Preferably, wherein R1 is a polymer moiety, R2 is selected from nil and a polymer moiety. Most preferably, wherein R1 is a polymer moiety, R2 is selected from nil and an identical polymer moiety. Wherein at least one of R1 and R2 are respectively, the first polymer and the second polymer, then X is preferably not nil.
Polypeptide Moieties
The polypeptide moieties described herein include those comprising two or more amino acid residues. Preferred polypeptide moieties are selected from proteins, including enzymes. Preferred enzymes include proteases, cellulases, lipases, amylases, peroxidases, microperoxidases, hemicellulases, xylanases, phospholipases, esterases, cutinases, pectinases, keratinases, reductases (including, for example, NADH reductase), oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, b-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccases, transferases, isomerases (including, for example, glucose isomerase and xylose isomerase), lyases, ligases, synthetases, and fruit-based enzymes (including, for example, papain). More preferred enzymes for use as polypeptide moieties include proteases, cellulases, amylases, lipases, and fruit-based enzymes, with proteases being even more preferred.
Examples of lipases for use as a polypeptide moiety include those derived from the following microorganisms: Humicola, Pseudonomas, Fusarium, Mucor, Chromobacterium, Aspergillus, Candida, Geotricum, Penicillium, Rhizopus, and Bacillus.
Examples of commercial lipases include Lipolase(copyright), Lipolase Ultra(copyright), Lipozyme(copyright), Palatase(copyright), Novozym435(copyright), and Lecitase(copyright) (all of which are commercially available from Novo Nordisk, Copenhagen, Denmark), Lumafast(copyright) (commercially available from Genencor, Int., Rochester, N.Y.), and Lipomax(copyright) (Genencor, Int.).
Examples of proteases for use as the polypeptide moiety include serine proteases, chymotrypsin, and elastase-type enzymes. The most preferred proteases for use as a polypeptide moiety include serine proteases, as were defined herein above in the discussion of xe2x80x9cprotease moietiesxe2x80x9d.
Most preferably, wherein the polypeptide moiety is a serine protease, the polypeptide moiety carries, independently, the definition of a protease moiety as described herein above. Preferably, as described above, the polypeptide moiety has a modified amino acid sequence of a parent amino acid sequence wherein the modification is in one or more of the clip site protection positions as described herein above (which parent amino acid sequence may be referred to as a xe2x80x9csecondxe2x80x9d parent amino acid sequence). In this instance, one of the linking moiety (wherein the linking moiety is not nil) or the protease moiety (wherein the linking moiety is nil) is covalently attached to the polypeptide moiety through one of the substituting amino acids present in one of the clip site protection positions of the polypeptide moiety. Wherein the polypeptide moiety is a serine protease, the same preferred, more preferred, and most preferred groupings of clip site protection positions apply as are described herein above for protease moieties and their corresponding parent amino acid sequences.
Most preferably, wherein the polypeptide moiety is a serine protease, the polypeptide moiety and the protease moiety are equivalent moieties. In this instance, the polypeptide moiety and the protease moiety are most preferably attached through a disulfide bridge, wherein X is nil, and most preferably, R2 is nil.
Polymer Moieties
The addition moieties herein may comprise a polymer moiety. As used herein, the term polymer moiety means any molecule which comprises two or more identical (preferably five or more identical) monomer units. Examples of suitable polymer moieties include polyalkylene oxides, polyalcohols, polyvinyl alcohols, polycarboxylates, polyvinylpyrrolidones, celluloses, dextrans, starches, glycogen, agaroses, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acid hydrosylates, and hydrosylates of chitosan. Preferred polyalkylene oxides include polyethylene glycols, methoxypolyethylene glycols, and polypropylene glycols. Preferred dextrans include carboxymethyldextrans. Preferred celluloses include methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethyl cellulose, carboxyethyl cellulose, and hydroxypropylcellulose. Preferred starches include hydroxyethyl starches and hydroxypropyl starches. The more preferred polymers are polyalkylene oxides. The most preferred polymer moiety is polyethylene glycol.
Wherein R1 and R2 are each, independently, polymer moieties, R1 and R2 preferably has a collective molecular weight (i.e., molecular weight of R1 plus molecular weight of R2) of from about 0.2 kD (kilodaltons) to about 40 kD, more preferably from about 0.5 kD to about 40 kD, even more preferably from about 0.5 kD to about 20 kD, and most preferably from about 1 kD to about 10 kD.
Wherein R1 and R2 are each polymer moieties, R1 and R2 each, independently, preferably have a molecular weight of about 0.1 kD to about 20 kD, more preferably from about 0.25 kD to about 20 kD, even more preferably from about 0.5 kD to about 10 kD, and most preferably from about 0.5 kD to about 5 kD.
Wherein R1 and R2 are each polymer moieties, the ratio of the molecular weights of R1 to R2 preferably ranges from about 1:10 to about 10:1, more preferably from about 1:5 to about 5:1, and most preferably from about 1:3 to about 3:1.
Wherein R1 is a polymer moiety and R2 is nil, R1 preferably has a molecular weight of from about 0.1 kD to about 40 kD, more preferably about 0.5 kD to about 40 kD, even more preferably from about 0.5 kD to about 20 kD, and most preferably from about 1 kD to about 10 kD.
Linking Moieties
As used herein, X may be nil or a linking moiety which is optionally covalently attached to one or more polypeptide moieties or one or more polymer moieties, or both, and is also covalently attached to an amino acid residue at one of the clip site protection positions of the protease moiety. The linking moiety may be, generally, any small molecule, i.e., a molecule having a molecular weight of less than about 1600, preferably less than about 800, more preferably less than about 400, and most preferably less than about 300. The most preferred linking moieties include those capable of being covalently bound to a cysteine residue or a lysine residue, most preferably a cysteine residue.
Examples of linking moieties and related chemistry are disclosed in U.S. Pat. No. 5,446,090, Harris, issued Aug. 29, 1995; U.S. Pat. No. 5,171,264, Merrill, issued Dec. 15, 1992; U.S. Pat. No. 5,162,430, Rhee et al., issued Nov. 10, 1992; U.S. Pat. No. 5,153,265, Shadle et al., issued Oct. 6, 1992; U.S. Pat. No. 5,122,614, Zalipsky, issued Jun. 16, 1992; Goodson et al., xe2x80x9cSite-Directed Pegylation of Recombinant Interleukin-2 at its Glycosylation Sitexe2x80x9d, Biotechnology, Vol. 8, No. 4, pp. 343-346 (1990); Kogan, xe2x80x9cThe Synthesis of Substituted Methoxy-Poly(ethylene glycol) Derivatives Suitable for Selective Protein Modificationxe2x80x9d, Synthetic Communications, Vol. 22, pp. 2417-2424 (1992); and Ishii et al., xe2x80x9cEffects of the State of the Succinimido-Ring on the Fluorescence and Structural Properties of Pyrene Maleimide-Labeled aa-Tropomyosinxe2x80x9d, Biophysical Journal, Vol. 50, pp. 75-80 (1986). The most preferred linking moiety is substituted (for example, alkyl) or unsubstituted succinimide.
As further examples, the following non-limiting reagents may be utilized to form the linking moiety: N-[alpha-maleimidoacetoxy]succinimide ester; N-5-azido-2-nitrobenzoyloxysuccinimide; bismaleimidohexane; N-[beta-maleimidopropyloxy]succinimide ester; bis[2-(succinimidyloxycarbonyloxy)-ethyl]sulfone; bis[sulfosuccinimidyl]suberate; 1,5-difluoro-2,4-dintrobenzene; dimethlyadipimate.2HCl; dimethylpimelimidate.2HCl; dimethylsuberimidate.2HCl; disuccinimidyl glutarate; disuccinimidyl suberate; m-maleimidobenzoyl-N-hydroxysuccinimide ester; N-hydroxysuccinimidyl-4-azidosalicylic acid; N-succinimidyl-6-[4xe2x80x2-azido-2xe2x80x2-nitrophenylamino]hexanoate; N-hydroxysuccinimidyl 2,3-dibromopropionate; succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate; succinimidyl 4-(p-maleimidophenyl)-butyrate; succinimidyl-6-[(beta-maleimidopropionamido)hexanoate]; bis[2-(sulfosuccinimidyloxycarbonyloxy)-ethyl]sulfone; N-[gamma-maleimidobutyryloxy]sulfosuccinimide ester; N-hydroxysulfosuccinimidyl-4-azidobenzoate; N-[kappa-maleimidoundecanoyloxy]sulfosuccinimide ester; m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester; sulfosuccinimidyl[4-azidosalicylamido]hexanoate; sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate; sulfosuccinimidyl 6-[4xe2x80x2-azido-2xe2x80x2-nitrophenylamino]hexanoate; sulfosuccinimidyl 4-[p-azidophenyl]butyrate; sulfosuccinimidyl[4-iodoacetyl]aminobenzoate; sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate; and sulfosuccinimidyl 4-(p-maleimidophenyl)-butyrate. Each of these reagents is commercially available from Pierce Chemical Co., Rockford, Ill.
Optional Moieties
The protease conjugate may additionally comprise one or more other chemical structures, including (for example) one or more small molecules, polypeptides, and/or polymers attached to other residues of the protease not herein exemplified or even at a clip site protection position not bearing an addition moiety (herein referred to as xe2x80x9csupplementary moietiesxe2x80x9d). Supplementary moieties may include polypeptide moieties, polymer moieties, and linking moieties as described herein above. Additionally, for example, one or more polymers (most preferably polyethylene glycol) having a molecular weight of from about 100 Da to about 5000 Da, preferably from about 100 Da to about 2000 Da, more preferably from about 100 Da to about 1000 Da, still more preferably from about 100 Da to about 750 Da, and most preferably about 300 Da may be covalently attached to the protease moiety herein at residues other than those exemplified herein. Such polymer moieties may be attached directly to the protease moiety herein, at any location of the protease moiety, using techniques as described herein and as well-known in the art (including through a linking moiety as described herein). Non-limiting examples of polymer conjugation of this optional type is set forth in WO 99/00849, Olsen et al., Novo Nordisk A/S, published Jan. 7, 1999.
The protease moieties having a substitution in one or more of the clip site protection positions (or any other location of the moiety) are prepared by mutating the nucleotide sequences that code for a parent amino acid sequence. Such methods are well-known in the art; a non-limiting example of one such method is set forth below:
A phagemid (pSS-5) containing the wild-type subtilisin BPNxe2x80x2 gene (Mitchison, C. and J. A. Wells, xe2x80x9cProtein Engineering of Disulfide Bonds in Subtilisin BPNxe2x80x2xe2x80x9d, Biochemistry, Vol. 28, pp. 480-4815 (1989) is transformed into Escherichia coli dut-ung-strain CJ236 and a single stranded uracil-containing DNA template is produced using the VCSM13 helper phage (Kunkel et al., xe2x80x9cRapid and Efficient Site-Specific Mutagenesis Without Phenotypic Selectionxe2x80x9d, Methods in Enzymology, Vol 154, pp. 367-382 (1987), as modified by Yuckenberg et al., xe2x80x9cSite-Directed in vitro Mutagenesis Using Uracil-Containing DNA and Phagemid Vectorsxe2x80x9d, Directed Mutagenesisxe2x80x94A Practical Approach, McPherson, M. J. ed., pp. 27-48 (1991). Primer site-directed mutagenesis modified from the method disclosed in Zoller, M. J., and M. Smith, xe2x80x9cOligonucleotidexe2x80x94Directed Mutagenesis Using M13xe2x80x94Derived Vectors: An Efficient and General Procedure for the Production of Point Mutations in any Fragment of DNAxe2x80x9d, Nucleic Acids Research, Vol. 10, pp. 6487-6500 (1982) is used to produce all mutants (essentially as presented by Yuckenberg et al., supra).
Oligonucleotides are made using a 380B DNA synthesizer (Applied Biosystems Inc.). Mutagenesis reaction products are transformed into Escherichia coli strain MM294 (American Type Culture Collection E. coli 33625). All mutations are confirmed by DNA sequencing and the isolated DNA is transformed into the Bacillus subtilis expression strain PG632 (Saunders et al., xe2x80x9cOptimization of the Signal-Sequence Cleavage Site for Secretion from Bacillus subtilis of a 34-Amino Acid Fragment of Human Parathyroid Hormonexe2x80x9d, Gene, Vol. 102, pp. 277-282 (1991) and Yang et al., xe2x80x9cCloning of the Neutral Protease Gene of Bacillus subtilis and the Use of the Cloned Gene to Create an in vitroxe2x80x94Derived Deletion Mutationxe2x80x9d, Journal of Bacteriology, Vol. 160, pp. 15-21 (1984).
Fermentation is as follows. Bacillus subtilis cells (PG632) containing the protease of interest are grown to mid-log phase in one liter of LB broth containing 10 g/L glucose, and inoculated into a Biostat C fermentor (Braun Biotech, Inc., Allentown, Pa.) in a total volume of 9 liters. The fermentation medium contains yeast extract, casein hydrosylate, solublexe2x80x94partially hydrolyzed starch (Maltrin M-250), antifoam, buffers, and trace minerals (see xe2x80x9cBiology of Bacilli: Applications to Industryxe2x80x9d, Doi, R. H. and M. McGloughlin, eds. (1992)). The broth is kept at a constant pH of 7.5 during the fermentation run. Kanamycin (50 xcexcg/mL) is added for antibiotic selection of the mutagenized plasmid. The cells are grown for 18 hours at 37xc2x0 C. to an A600 of about 60 and the product harvested.
The fermentation broth is taken through the following steps to obtain pure protease. The broth is cleared of Bacillus subtilis cells by tangential flow against a 0.16 xcexcm membrane. The cell-free broth is then concentrated by ultrafiltration with a 8,000 molecular weight cut-off membrane. The pH is adjusted to 5.5 with concentrated MES buffer (2-(N-morpholino)ethanesulfonic acid). The protease is further purified by cation exchange chromatography with S-sepharose and elution with NaCl gradients. See Scopes, R. K., xe2x80x9cProtein Purification Principles and Practicexe2x80x9d, Springer-Verlag, New York (1984)
A pNA assay (DelMar et al., Analytical Biochemistry, Vol. 99, pp. 316-320 (1979)) is used to determine the active protease concentration for fractions collected during gradient elution. This assay measures the rate at which p-nitroaniline is released as the protease hydrolyzes the soluble synthetic substrate, succinyl-alanine-alanine-proline-phenylalanine-p-nitroaniline (sAAPF-pNA). The rate of production of yellow color from the hydrolysis reaction is measured at 410 nm on a spectrophotometer and is proportional to the active protease moiety concentration. In addition, absorbance measurements at 280 nm are used to determine the total protein concentration. The active protease/total-protein ratio gives the protease purity, and is used to identify fractions to be pooled for the stock solution.
To avoid autolysis of the protease during storage, an equal weight of propylene glycol is added to the pooled fractions obtained from the chromatography column. Upon completion of the purification procedure the purity of the stock protease solution is checked with SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and the absolute enzyme concentration is determined via an active site titration method using trypsin inhibitor type II-T: turkey egg white (Sigma Chemical Company, St. Louis, Mo.).
In preparation for use, the protease stock solution is eluted through a Sephadex-G25 (Pharmacia, Piscataway, N.J.) size exclusion column to remove the propylene glycol and exchange the buffer. The MES buffer in the enzyme stock solution is exchanged for 0.01 M KH2PO4 solution, pH 5.5.
With the protease prepared it may be utilized for functionalization with one or more addition moieties to produce the protease conjugate. The precursor to the addition moiety (the precursor to the addition moiety reacts with the precursor to the protease moiety to form the protease conjugate which is comprised of the addition moiety and the protease moiety) is preferably activated to enhance reactivity with the precursor to the protease moiety. Such activation is well-known in the art. Non-limiting examples of methods of protease conjugate preparation are provided below.